Progress in several areas of medicine (i.e. mental health, cardiology, immunity etc.), where new and more effective drugs are needed, is severely impeded by the extreme cost engendered by undesired drug-induced adverse effects associated with several candidate lead compounds in the drug discovery pipeline. One major drawback of the current drug discovery process, for all drug development, is the high rate of attrition of lead compounds caused by unforeseen adverse drug effects, notably cardiotoxic effects, often detected in the later rather than in the earlier phases of the drug discovery pipeline. The prevention of drug-induced cardiotoxicity, which may manifest itself as cardiac arrhythmias, represents the highest priority for regulatory agencies and (bio)pharmaceutical companies, since manifestation of this type of toxicity is immediately life-threatening. It has been shown that 33% of adverse safety events in clinical studies are generally attributed to cardiac arrhythmic effects, which may lead to sudden death or severe cardiac complications in subjects (Mordwinkin et al (2013) Journal of cardiovascular translational research, Vol: 6(1):22-30).
Therefore, there is an urgent need for new cost-effective strategies to improve the traditional process of drug development/discovery, e.g. eliminate drug-induced cardiotoxic effects. Particularly, there is an urgent unmet need for predicting drug-induced cardiotoxicity at an early stage in the drug pipeline development. Over the last decade, considerable research efforts have been devoted towards this goal. For instance, several biological models and tools have been developed including the use of pluripotent stem cells, ion-channel assays, and computational tools. Particularly, the use of cardiomyocytes derived from pluripotent stem cells is a current focus of interest in the development of innovative predictive assays to rectify the issues relating to cardiotoxicity during drug development.
Pluripotent stem cells, such as pluripotent embryonic stem cells (PESC) and induced pluripotent stem cells (iPSC), are a potential source of cells for generating cardiomyocytes in in vitro culture. The use of cardiomyocytes is not only important for the development of assays for predicting drug-induced toxicity for all drugs in development, but is also important for cardiac research as well as for the development of new cardiac drugs in general, where cardiomyocytes can be used to uncover new drug targets and assess cardiac drug safety. The ability to produce cardiomyocytes from PESC and/or iPSC in vitro also opens other therapeutic avenues such as regenerative medicine, where PESC- and/or iPSC-derived cardiomyocytes can be used to directly repair damaged cardiac tissues in patents in need thereof, for instance.
The ability to use cardiomyocytes in drug development/discovery, drug safety assay, cardiac disease modelling, cardiac research, regenerative medicine and other biological purposes largely depends on the ability to cultivate and obtain cardiomyocytes derived from PESC and/or iPSC in culture in vitro, which must meet certain phenotypic requirements such as, for instance, a certain level of functional properties (e.g. adult-like electrophysiological patterns) and/or genetic profile (e.g. adult-like expression of cellular fate-specific genetic markers), and/or morphological aspects. (e.g. adult-like shape and histological properties).
In this respect, a main limitation of the art is that current methods and culture medium compositions for generating cardiomyocytes from PESC and/or iPSC in in vitro culture yield results which are suboptimal and do not or only in part match the requirements for applications such as drug development/discovery, drug safety assay, cardiac disease modelling, cardiac research, regenerative medicine, where cardiomyocytes suitable for ‘real-life’ context (i.e. adult-like state) are needed. That is because current methods and culture medium compositions yield immature cardiomyocytes, which are akin to foetal (foetal-like) cardiomyocytes. Such cardiomyocytes are inadequate for use in applications such as drug development/discovery, drug safety assay, cardiac disease modelling, cardiac research, regenerative medicine and other purposes aimed to model or treat cardiac conditions that typically occur in adulthood and not at earlier developmental stages. Hence, adult-like (mature) cardiomyocytes are better suited than immature cardiomyocytes or foetal-like cardiomyocytes for these purposes.
Therefore, there is a need for improved methods and culture medium compositions for generating adult-like cardiomyocytes (as opposed to immature cardiomyocytes) from PESC and/or iPSC in in vitro culture. There is also a need for improved methods and culture medium compositions to produce larger amount (greater yield) of adult-like cardiomyocytes derived from foetal-like (immature) cardiomyocytes, which are themselves derived from PESC and/or iPSC in in vitro differentiation of the stem cells.
It is an object of the present invention to overcome the major limitations of the art by providing chemically-defined culture medium compositions that promote maturation of foetal-like (immature) cardiomyocytes, which are themselves derived from pluripotent embryonic stem cells in in vitro culture and methods for generating said adult-like cardiomyocytes in in vitro culture, which are more efficient and support larger scale production of adult-like cardiomyocytes that meet the general requirements for cardiac research and clinical applications.